Antisense oligomers and methods for inducing immune tolerance and immunosuppression

ABSTRACT

A method and composition for inducing human dendritic cells to a condition of reduced capacity for antigen-specific activation of T cells, and, in mature dendritic cells, increased production of extracellular IL-10 is disclosed. A population of dendritic cells is exposed to a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to an expression-sensitive region of a preprocessed or processed human CD86 transcript identified, in its processed form, by SEQ ID NO:33, to form a duplex structure between said compound and transcript having a Tm of at least 45° C. Formation of the duplex blocks expression of full-length CD86 in said cells, which in turn leads to reduced capacity for antigen-specific activation of T cells, and, in mature dendritic cells, increased production of extracellular IL-10.

This application claims priority to U.S. Provisional Application No.60,538,655, filed Jan. 23, 2004, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compounds and methods of inducingimmunological tolerance using a peptide-antisense conjugate toselectively limit costimulation of naïve T-cells by mature dendriticcells and formation of a cytokine microenvironment that augmentstolerized T-cells.

REFERENCES

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BACKGROUND OF THE INVENTION

Transplantation of allogeneic donor cells, tissues or organs(transplantation between genetically different individuals of the samespecies) is used to treat a variety of conditions (typically tissue, ororgan-failure conditions) and is often the sole or highly preferredtherapeutic option. The list of successfully transplanted cells, tissuesand organs includes kidney, heart, lung, liver, corneas, pancreas,marrow, skin, and bones. However, allogeneic transplantation involvessignificant risks and drawbacks, including graft rejection,complications from immunosuppressive therapy and graft-versus hostdisease which are frequently highly debilitating or lethal.

Rejection of allografts is presently understood to be initiated by therecognition of allogeneic (i.e. donor) major histocompatibility complex(MHC) molecules by recipient T-lymphocytes, leading to upregulatedcellular and humoral immunity through activation of T cells. The MHCantigens are typically presented to the recipient T-lymphocytes byantigen presenting cells, such as macrophages and dendritic cells.Although immunosuppressive drugs such as cyclosporine may be used in anattempt to modulate rejection, these immunosuppressive agents havesevere side effects and often fail to prevent continued rejectionepisodes.

Dendritric cells (DCs) are a family of professional antigen presentingcells (APCs) that are present in virtually all tissues of the body. Theability of dendritic cells to capture foreign antigens, migrate tolymphoid tissues and redistribute antigen-MHC to the cell surface alongwith appropriate costimulatory signals are well known T-cell primingfunctions for these APCs. In addition to these immunostimulatoryproperties, dendritic cells are also known to play a role indown-regulating immune responses. Certain subpopulations of dendriticcells, acting as professional APCs, also maintain and regulate T-celltolerance in the periphery. There is thus a need for therapeutic methodsand compositions capable of inducing immunological tolerance with lowertoxicity and improved efficacy.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, a method of inducing humandendritic cells to a condition of reduced capacity for antigen-specificactivation of T cells, and, in mature dendritic cells, increasedproduction of extracellular IL-10. The method includes exposing apopulation of human dendritic cells to a substantially unchargedantisense compound containing 12-40 subunits and a base sequenceeffective to hybridize to an expression-sensitive region of apreprocessed or processed human CD-86 transcript identified, in itsprocessed form, by SEQ ID NO:33, to form, between the compound andtranscript, a heteroduplex structure having a Tm of at least 45° C. Theheteroduplex formation blocks expression of full-length CD86 in thecells, which in turn, produces inhibition of the expression offull-length CD86 on the surface of dendritic cells, and producesenhanced expression of extracellular IL-10 by mature dendritic cells.

In a preferred embodiment, the antisense compound to which the dendriticcells are exposed is composed of phosphorus-containing intersubunitlinkages joining a morpholino nitrogen of one subunit to a 5′ exocycliccarbon of an adjacent subunit. In an exemplary compound, the morpholinosubunits in the compound are joined by phosphorodiamidate linkages, inaccordance with the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkylamino, and the heteroduplex structure formed has a Tm of at least 50° C.For example, X=NR₂, where each R is independently hydrogen or methyl inthe compound to which the dendritic cells are exposed.

The compound may be covalently linked, at one compound end, to anarginine-rich peptide effective to enhance uptake of the compound intothe dendritic cells. Exemplary arginine-rich peptides are those havingthe sequences SEQ ID NOS: 1 or 2. Where the dendritic cells include amixture of immature and mature dendritic cells, the arginine-richpeptide may be an rTAT peptide having the sequence identified by SEQ IDNO: 1. This peptide is effective to achieve a greater level ofintracellular uptake of the antisense compound into the mature dendriticcells than is achieved (i) in the immature dendritic cells, or (ii) byexposing the mature dendritic cells to the antisense compound in theabsence of the rTAT polypeptide.

More generally, the rTAT peptide may be coupled to any antisense orother therapeutic compound to achieve selective uptake of the compoundinto mature dendritic cells, relative to uptake in immature cells.

Where the antisense compound is effective to hybridize to anexpression-sensitive target region adjacent the start site of theprocessed human CD86 transcript, the compound may have a base sequencethat is complementary to a target region containing at least 12contiguous bases in a processed human CD86 transcript identified by SEQID NO:9, where the compound is effective to block translation of theprocessed transcript. The antisense compound may have, for example, oneof the base sequence identified by SEQ ID NOS:21-23 and 32.

Where the antisense compound is effective to hybridize to a splice siteof preprocessed human CD86, the compound may have a base sequence thatis complementary to at least 12 contiguous bases of a splice site in apreprocessed human CD86 transcript, where the compound is effective toblock processing of a preprocessed CD86 transcript to produce afull-length, processed CD 86 transcript. The splice site in thepreprocessed CD86 transcript may have one of the sequences identified bySEQ ID NOS:10-14. The antisense compound may have, for example, one ofthe base sequences identified by SEQ ID NOS:24-28.

For use in inhibiting transplantation rejection in a human subjectreceiving an allograft tissue or organ, the compound is administered tothe subject in an amount effective to inhibit the rate and extent ofrejection of the transplant. The compound may be administered both priorto and following the allograft tissue or organ transplantation in thesubject, and compound administration may be carried out for a selectedperiod of 1-3 weeks. The compound may be further administered to thesubject, as needed, to control the extent of transplantation rejectionin the subject.

For use in treating an autoimmune condition in a human subject, thecompound may be administered to the subject, in an amount effective toreduce the severity of the autoimmune condition. The compound may beadministered over an extended period of time, as needed, to control theseverity of the autoimmune condition in the subject.

In another aspect, the invention provides a composition for use ininducing dendritic cells to a condition of reduced capacity forantigen-specific activation of T cells, and, in mature dendritic cells,increased production of extracellular IL-10. The compound comprises asubstantially uncharged antisense compound containing 12-40 subunits anda base sequence effective to hybridize to an expression-sensitive regionof a preprocessed or processed human CD-86 transcript identified, in itsprocessed form, by SEQ ID NO:33, to form a heteroduplex structurebetween said compound and transcript having a Tm of at least 45° C.Exemplary features of the compound are as described above.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-D show several preferred morpholino-type subunits having 5-atom(A), six-atom (B) and seven-atom (C-D) linking groups suitable forforming polymers.

FIGS. 2A-D show the repeating subunit segment of exemplary morpholinooligonucleotides, designated A through D, constructed using subunitsA-D, respectively, of FIG. 1.

FIGS. 3A-3G show examples of uncharged linkage types in oligonucleotideanalogs.

FIG. 4 shows the chemical structures of a phosphorodiamidate morpholinooligomer conjugates and the sequences of peptide conjugates used in thisinvention. Fluorescein can be linked to the 3′ end of the peptide-PMOconjugate to allow imaging and/or detection of PMO uptake in intactcells.

FIG. 5 shows a fluorescence activated cell sorting (FACS) analysis ofthe uptake of fluorescein-labeled peptide-PMO conjugates into dendriticcells subjected to lipopolysaccharide (LPS) activation.

FIG. 6 shows antisense PMO to CD86 inhibits expression of both CD86 andCD80 in dendritic cells.

FIG. 7 demonstrates that blocking CD86 interactions does not lead toIL-10 induction in dendritic cells.

FIGS. 8A and 8B shows that antisense PMO targeting of splice donor oracceptor sites alters CD86 mRNA.

FIG. 9 demonstrates that antisense PMO targeted to the CD86 start codonor Exon 10 of the CD86 gene alters the morphology of thelipopolysaccharide-treated dendritic cells.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise.

The terms “CD80” and “CD86” refer to the costimulatory protein moleculesthat are expressed on the surface of mature, antigen presentingdendritic cells. T cell activation is dependent upon signals deliveredthrough the antigen-specific T cell receptor and accessory costimulatoryreceptors on the T cell. The CD28 costimulatory receptor isconstitutively expressed on T cells. Engagement of CD28 on naïve T cellsby either CD80 or CD86 ligands on antigen presenting cells (e.g. maturedendritic cells) provides a potent costimulatory signal to T cellsactivated through their T cell receptor. The CD80 and CD86 costimulatorymolecules are also known as B7-1 and B7-2, respectively. The term “B7molecules” refer collectively to the CD80 and CD86 molecules.

The term “antigen-activated T cells” refers to T cells that becomeactivated after the T cell receptor (TCR) complex and a co-stimulatoryreceptor (e.g. CD28 on naïve CD4 and CD8 T cells) are engaged to theextent that a signal transduction cascade is initiated. Antigen is boundby the TCR in the form of a foreign peptide in the context of a self MHCmolecule, either Class I or Class II, in the case of CD4 and CD8 T cellsrespectively, conferring the antigen specificity of the T cell. Uponactivation, T cells will proliferate and then secrete cytokines or carryout cytolysis on cells expressing the foreign peptide with self MHC.Cytokines are growth factors for other T cells or signals for B cells toproduce antibody.

The term “antigen-activated B cells” refer to either of two differenttypes of B cell activation, T cell dependent and T cell independent. Tcell independent antigens contain repetitive identical epitopes and arecapable of clustering membrane bound antibody on the surface of the Bcell which can result in delivering activation signals. T cell dependentactivation is in response to protein antigens where the B cell acts as aprofessional antigen presenting cell. Surface antibody bound to antigenis internalized by the B cell, the antigen processed and presented aspeptides on the B cell surface bound to MHC II molecules. Responding Tcells recognize the peptide as foreign in the context of self MHC andrespond by secreting cytokines and expression of CD40L. Together theseprovide a co-stimulatory signal to the B cell. In either case of B cellactivation the cell will proliferate and differentiate into plasma Bcells capable of secreting antibodies against the antigen.

The terms “activated dendritic cells” and “mature dendritic cells” (DCs)refer to professional antigen-presenting cells (APCs) capable ofexpressing both MHC class I and II and co-stimulatory moleculesincluding CD80 (B7-1) and CD86 (B7-2). Two different DC phenotypes areexhibited depending on maturation state and location in the body.Immature DCs reside in all tissues and organs as active phagocyticcells. Mature DCs traffic to secondary lymphoid organs (e.g. lymph nodeand spleen) and present peptides derived from processed protein antigensto T cells in the context of MHC molecules. Mature DCs also provide thenecessary co-stimulatory signals to T cells by expressing theappropriate surface ligand (e.g. CD80 and CD86 on DCs bind to CD28 on Tcells).

The terms “antisense oligonucleotides,” “antisense oligomer,” and“antisense compound” are used interchangeably and refer to a compoundhaving a sequence of nucleotide bases and a subunit-to-subunit backbonethat allows the antisense oligomer to hybridize to a target sequence inan RNA by Watson-Crick base pairing, to form an RNA:oligomer heterduplexwithin the target sequence. The antisense oligonucleotide includes asequence of purine and pyrimidine heterocyclic bases, supported by abackbone, which are effective to hydrogen-bond to corresponding,contiguous bases in a target nucleic acid sequence. The backbone iscomposed of subunit backbone moieties supporting the purine andpyrimidine heterocyclic bases at positions that allow such hydrogenbonding. These backbone moieties are cyclic moieties of 5 to 7 atoms inlength, linked together by phosphorous-containing linkages one to threeatoms long.

A “morpholino” oligonucleotide refers to a polymeric molecule having abackbone which supports bases capable of hydrogen bonding to typicalpolynucleotides, wherein the polymer lacks a pentose sugar backbonemoiety, and more specifically a ribose backbone linked by phosphodiesterbonds which is typical of nucleotides and nucleosides, but insteadcontains a ring nitrogen with coupling through the ring nitrogen. Apreferred “morpholino” oligonucleotide is composed of morpholino subunitstructures of the form shown in FIG. 1A-1D, where (i) the structures arelinked together by phosphorous-containing linkages, one to three atomslong, joining the morpholino nitrogen of one subunit to the 5′ exocycliccarbon of an adjacent subunit, and (ii) Pi and Pj are purine orpyrimidine base-pairing moieties effective to bind, by base-specifichydrogen bonding, to a base in a polynucleotide. Exemplary structuresfor antisense oligonucleotides for use in the invention include themorpholino subunit types shown in FIGS. 1A-1D, with the uncharged,phosphorous-containing linkages shown in FIGS. 2A-2D, and moregenerally, the uncharged linkages 3A-3G.

As used herein, an oligonucleotide or antisense oligomer “specificallyhybridizes” to a target polynucleotide if the oligomer hybridizes to thetarget under physiological conditions, with a thermal melting point (Tm)substantially greater than 37° C., preferably at least 45° C., andtypically 50° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions, selected to be about10° C., and preferably about 50° C. lower than the Tm for the specificsequence at a defined ionic strength and pH. At a given ionic strengthand pH, the Tm is the temperature at which 50% of a target sequencehybridizes to a complementary polynucleotide.

Polynucleotides are described as “complementary” to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. A double-stranded polynucleotide can be“complementary” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second.Complementarity (the degree that one polynucleotide is complementarywith another) is quantifiable in terms of the proportion of bases inopposing strands that are expected to form hydrogen bonds with eachother, according to generally accepted base-pairing rules. An antisensecompound may be complementary to a target region of a target transcripteven if the two bases sequences are not 100% complementary, as long asthe heteroduplex structure formed between the compound and transcripthas the desired Tm stability.

As used herein the term “analog” with reference to an oligomer means asubstance possessing both structural and chemical properties similar tothose of the reference oligomer.

As used herein, a first sequence is an “antisense sequence” or“targeting sequence” with respect to a second sequence or “targetsequence” if a polynucleotide whose sequence is the first sequencespecifically binds to, or specifically hybridizes with, the secondpolynucleotide sequence under physiological conditions.

As used herein, “effective amount” relative to an antisense oligomerrefers to the amount of antisense oligomer administered to a subject,either as a single dose or as part of a series of doses, that iseffective to inhibit expression of a selected target nucleic acidsequence.

As used herein, an “expression-sensitive region” of a processed orpreprocessed mRNA transcript refers to either (i) a region including oradjacent the AUG start site of a processed transcript, where formationof an antisense-transcript heteroduplex is effective to inhibittranslation of the transcript or (ii) a region including or adjacent adonor or acceptor splice site junction in a preprocessed transcript,where formation of an antisense-transcript heteroduplex is effective toinhibit formation of a full-length processed transcript, either becauseone or more exons that would normally be included in the transcript havebeen deleted or because the transcript has been truncated at the targetsplice site.

“Dendritic cells” are specialized antigen presenting cells (APCS) withpotent capacity to initiate and direct the antigen-specific responses ofnaïve T cells. This heterogeneous population of cells reside in bloodand all tissues as two phenotypically and functionally distinct forms.“Immature DCs” are highly phagocytic, proficient for antigen processingand characterized by low-level expression of major histocompatibilitycomplex (MHC) class II and T cell costimulatory ligands of the B7family, CD80 and CD86. Maturation can be triggered by various stimuliderived from either host or pathogen. In response to such stimuli,“mature DCs” cease phagocytic activity and significantly increasesurface expression of MHC II, CD80 and CD86. Consequently mature DCs arecapable of providing sufficient ligand to trigger T cell activationthrough the T cell and costimulatory receptors.

Abbreviations:

-   -   PMO=phosphorodiamidate morpholino oligomer    -   AICD=activation induced cell death    -   MHC=major histocompatibility    -   TCR=T cell receptor    -   DC=dendritic cell    -   APC=antigen presenting cell        II. The Role of Antigen Presenting Cells in Transplantation and        Autoimmune Disorders

There is evidence from the two signal model for T cell activation thatcostimulation by the engagement of dendritic-cell CD86 molecules withCD28 on T cells is necessary for the complete induction of T cellresponses. The process of antigen presentation whereby MHC plus peptideantigen are presented by the dendritic cell to the T cell receptor (TCR)is termed “signal 1”. In circumstances where there is eitherinsufficient or an absence of costimulation (termed “signal 2”) duringthe process of antigen presentation, antigen specific tolerance canoccur.

Tolerance produced by a loss in signal 2 can be a result of clonaldeletion (T cell death in the population of cells recognizing signal 1),anergy (non-responsiveness in the antigen-specific population onsubsequent encounters) or through the generation of a regulatorypopulation of antigen-specific T cells. Regulatory T cells can beinduced through the presentation of antigen by immature dendritic cellswhich express reduced levels of CD86 compared to mature dendritic cells.Regulatory T cells provide a form of functional tolerance whereby theyproduce inhibitory cytokines (IL-4, IL-10, and TGF-beta) whenencountering antigen and thus inhibit the organism from responding tothe antigen (i.e., immuno-suppression). It has been shown that theprocess of generating T regulatory cells can be further facilitated byproviding a third signal (“signal 3”) that is the type of cytokinesproduced by the antigen presenting cell while providing signal 1 and 2.

IL-10, previously termed “cytokine synthesis inhibiting factor” due toits ability to inhibit cytokine production of most immune cell types,can provide a signal 3 to T cells. During the early stages of T cellactivation, upon responding to antigen, the cytokines produced by thedendritic cell presenting antigen will promote the resulting phenotypeof the responding T cell. IL-12 and IL-4 produced by dendritic cellspromote T cell responses of the Th1 and Th2 phenotypes, respectively.The T cell types in turn direct the production of cytotoxic T cells orantibodies, respectively, which are effector cells and molecules capableof rejecting transplants or producing autoimmune disease. Th3 T cellsexhibit a regulatory phenotype that directly inhibits the development ofany future T cell responses from becoming either a Th1 or Th2 cellcapable of responding to the antigen recognized by the Th3 cell or thenewly responding T cells. Dendritic cells producing IL-10 can thusinduce a tolergenic response by diverting the development of T cellresponses from Th1 and Th2 to a Th3 T cell type.

The present invention provides a means to precisely and specificallyalter the manner in which dendritic cells elicit antigen-specific immuneresponses from T cells. In particular a diminution in the level of CD86protein is achieved by antisense inhibition targeted to dendritic cells.Experiments conducted in support of the invention demonstrated thatmaturing DCs produce increased amounts of IL-10 as a result ofdiminished CD86 expression. Moreover, it was determined that thecytoplasmic region encoded by exon 10 is functionally linked to theregulation of this cytokine.

III. Antisense Compound for Targeting Activated Immune Cells

A. Antisense Compound

Antisense oligomers for use in practicing the invention preferably havethe properties: (1) a backbone that is substantially uncharged, (2) theability to hybridize with the complementary sequence of a target RNAwith high affinity, that is a Tm substantially greater than 37° C.,preferably at least 45° C., and typically greater than 50° C., e.g., 60°C.-80° C. or higher, (3) a subunit length of at least 8 bases, generallyabout 8-40 bases, preferably 12-25 bases, and (4) nuclease resistance.In addition, the antisense compound may have the capability for activeor facilitated transport as evidenced by (i) competitive binding with aphosphorothioate antisense oligomer, and/or (ii) the ability totransport a detectable reporter into target cells.

Candidate antisense oligomers may be evaluated, according to well knownmethods, for acute and chronic cellular toxicity, such as the effect onprotein and DNA synthesis as measured via incorporation of 3H-leucineand 3H-thymidine, respectively. In addition, various controloligonucleotides, e.g., control oligonucleotides such as sense, nonsenseor scrambled antisense sequences, or sequences containing mismatchedbases, in order to confirm the specificity of binding of candidateantisense oligomers. The outcome of such tests is important indiscerning specific effects of antisense inhibition of gene expressionfrom indiscriminate suppression. Accordingly, sequences may be modifiedas needed to limit non-specific binding of antisense oligomers tonon-target nucleic acid sequences.

Heteroduplex formation. The effectiveness of a given antisense oligomermolecule in forming a heteroduplex with the target mRNA may bedetermined by screening methods known in the art. For example, theoligomer is incubated in a cell culture containing an mRNApreferentially expressed in activated lymphocytes, and the effect on thetarget mRNA is evaluated by monitoring the presence or absence of (1)heteroduplex formation with the target sequence and non-target sequencesusing procedures known to those of skill in the art, (2) the amount ofthe target mRNA expressed by activated lymphocytes, as determined bystandard techniques such as RT-PCR or Northern blot, (3) the amount ofprotein transcribed from the target mRNA, as determined by standardtechniques such as ELISA or Western blotting. (See, for example, (Pari,Field et al. 1995; Anderson, Fox et al. 1996). For the purposes of theinvention, a preferred test for the effectiveness of the CD86 antisenseoligomer is by measuring the induction of IL-10 expression and loss ofCD86 expression in mature dendritic cells treated with a CD86 PMOantisense compound.

Uptake into cells. A second test measures cell transport, by examiningthe ability of the test compound to transport a labeled reporter, e.g.,a fluorescence reporter, into cells. The cells are incubated in thepresence of labeled test compound, added at a final concentrationbetween about 10-300 nM. After incubation for 30-120 minutes, the cellsare examined, e.g., by microscopy or FACS analysis, for intracellularlabel. The presence of significant intracellular label is evidence thatthe test compound is transported by facilitated or active transport.

In one embodiment of the invention, uptake into cells is enhanced byadministering the antisense compound in combination with anarginine-rich peptide linked to the 5′ or 3′ end of the antisenseoligomer. The peptide is typically 8-16 amino acids and consists of amixture of arginine, and other amino acids including phenylalanine andcysteine, as discussed further below.

RNAse resistance. Two general mechanisms have been proposed to accountfor inhibition of expression by antisense oligonucleotides (Agrawal,Mayrand et al. 1990; Bonham, Brown et al. 1995; Boudvillain, Guerin etal. 1997). In the first, a heteroduplex formed between theoligonucleotide and the viral RNA acts as a substrate for RNaseH,leading to cleavage of the RNA. Oligonucleotides belonging, or proposedto belong, to this class include phosphorothioates, phosphotriesters,and phosphodiesters (unmodified “natural” oligonucleotides). Suchcompounds expose the RNA in an oligomer:RNA duplex structure tohydrolysis by RNaseH, and therefore loss of function.

A second class of oligonucleotide analogs, termed “steric blockers” or,alternatively, “RNaseH inactive” or “RNaseH resistant”, have not beenobserved to act as a substrate for RNaseH, and act by stericallyblocking target RNA nucleocytoplasmic transport, splicing, translation,or replication. This class includes methylphosphonates (Toulme, Tinevezet al. 1996), morpholino oligonucleotides, peptide nucleic acids(PNA's), certain 2′-O-allyl or 2′-O-alkyl modified oligonucleotides(Bonham, Brown et al. 1995), and N3′→P5′ phosphoramidates (Ding,Grayaznov et al. 1996; Gee, Robbins et al. 1998).

A test oligomer can be assayed for its RNaseH resistance by forming anRNA:oligomer duplex with the test compound, then incubating the duplexwith RNaseH under a standard assay conditions, as described (Stein,Foster et al. 1997). After exposure to RNaseH, the presence or absenceof intact duplex can be monitored by gel electrophoresis or massspectrometry.

In vivo uptake. In accordance with another aspect of the invention,there is provided a simple, rapid test for confirming that a givenantisense oligomer type provides the required characteristics notedabove, namely, high Tm, ability to be actively taken up by the hostcells, and substantial resistance to RNaseH. This method is based on thediscovery that a properly designed antisense compound will form a stableheteroduplex with the complementary portion of the RNA target whenadministered to a mammalian subject, and the heteroduplex subsequentlyappears in the urine (or other body fluid). Details of this method arealso given in co-owned U.S. Pat. No. 6,365,351 for “Non-Invasive Methodfor Detecting Target RNA,” the disclosure of which is incorporatedherein by reference.

Briefly, a test oligomer containing a backbone to be evaluated, having abase sequence targeted against a known RNA, is injected into a mammaliansubject. The antisense oligomer may be directed against anyintracellular RNA, including RNA encoded by a host gene. Several hours(typically 8-72) after administration, the urine is assayed for thepresence of the antisense-RNA heteroduplex. If heteroduplex is detected,the backbone is suitable for use in the antisense oligomers of thepresent invention.

The test oligomer may be labeled, e.g. by a fluorescent or a radioactivetag, to facilitate subsequent analyses, if it is appropriate for themammalian subject. The assay can be in any suitable solid-phase or fluidformat. Generally, a solid-phase assay involves first binding theheteroduplex analyte to a solid-phase support, e.g., particles or apolymer or test-strip substrate, and detecting the presence/amount ofheteroduplex bound. In a fluid-phase assay, the analyte sample istypically pretreated to remove interfering sample components. If theoligomer is labeled, the presence of the heteroduplex is confirmed bydetecting the label tags. For non-labeled compounds, the heteroduplexmay be detected by immunoassay if in solid phase format or by massspectroscopy or other known methods if in solution or suspension format.

Structural features. As detailed above, the antisense oligomer has abase sequence directed to a targeted portion of a cellular gene,preferably the region at or adjacent the start codon or a processedtranscript or a region at or adjacent a splice site junction of the CD86mRNA or preprocessed transcript. In addition, the oligomer is able toeffectively inhibit expression of the targeted gene when administered toa host cell, e.g. in a mammalian subject. This requirement is met whenthe oligomer compound (a) has the ability to be taken up by dendriticcells and (b) once taken up, form a duplex with the target RNA with a Tmgreater than about 45° C., preferably greater than 50° C.

The ability to be taken up selectively by activated immune cellsrequires, in part, that the oligomer backbone be substantiallyuncharged. The ability of the oligomer to form a stable duplex with thetarget RNA will depend on the oligomer backbone, the length and degreeof complementarity of the antisense oligomer with respect to the target,the ratio of G:C to A:T base matches, and the positions of anymismatched bases. The ability of the antisense oligomer to resistcellular nucleases promotes survival and ultimate delivery of the agentto the cell cytoplasm.

Antisense oligonucleotides of 15-20 bases are generally long enough tohave one complementary sequence in the mammalian genome. In addition,antisense compounds having a length of at least 12, typically at least15 nucleotides in length hybridize well with their target mRNA. Due totheir hydrophobicity, antisense oligonucleotides tend to interact wellwith phospholipid membranes, and it has been suggested that followingthe interaction with the cellular plasma membrane, oligonucleotides areactively transported into living cells (Loke, Stein et al. 1989;Yakubov, Deeva et al. 1989; Anderson, Xiong et al. 1999).

Morpholino oligonucleotides, particularly phosphoramidate- orphosphorodiamidate-linked morpholino oligonucleotides have been shown tohave high binding affinities for complementary or near-complementarynucleic acids. Morpholino oligomers also exhibit little or nonon-specific antisense activity, afford good water solubility, areimmune to nucleases, and are designed to have low production costs(Summerton and Weller 1997).

Morpholino oligonucleotides (including antisense oligomers) aredetailed, for example, in co-owned U.S. Pat. Nos. 5,698,685, 5,217,866,5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521,063, and 5,506,337,all of which are expressly incorporated by reference herein In onepreferred approach, antisense oligomers for use in practicing theinvention are composed of morpholino subunits of the form shown in theabove cited patents, where (i) the morpholino groups are linked togetherby uncharged linkages, one to three atoms long, joining the morpholinonitrogen of one subunit to the 5′ exocyclic carbon of an adjacentsubunit, and (ii) the base attached to the morpholino group is a purineor pyrimidine base-pairing moiety effective to bind, by base-specifichydrogen bonding, to a base in a polynucleotide. The purine orpyrimidine base-pairing moiety is typically adenine, cytosine, guanine,uracil or thymine. Preparation of such oligomers is described in detailin U.S. Pat. No. 5,185,444 (Summerton et al., 1993), which is herebyincorporated by reference in its entirety. As shown in this reference,several types of nonionic linkages may be used to construct a morpholinobackbone.

Exemplary subunit structures for antisense oligonucleotides of theinvention include the morpholino subunit types shown in FIGS. 1A-D, eachlinked by an uncharged, phosphorous-containing subunit linkage, as shownin FIGS. 2A-2D, respectively. In these figures, the X moiety pendantfrom the phosphorous may be any of the following: fluorine; an alkyl orsubstituted alkyl; an alkoxy or substituted alkoxy; a thioalkoxy orsubstituted thioalkoxy; or, an unsubstituted, monosubstituted, ordisubstituted nitrogen, including cyclic structures. Alkyl, alkoxy andthioalkoxy preferably include 1-6 carbon atoms, and more preferably 1-4carbon atoms. Monosubstituted or disubstituted nitrogen preferablyrefers to lower alkyl substitution, and the cyclic structures arepreferably 5- to 7-membered nitrogen heterocycles optionally containing1-2 additional heteroatoms selected from oxygen, nitrogen, and sulfur. Zis sulfur or oxygen, and is preferably oxygen.

FIG. 1A shows a phosphorous-containing linkage which forms the five atomrepeating-unit backbone shown in FIG. 2A, where the morpholino rings arelinked by a 1-atom phosphoamide linkage. Subunit B in FIG. 1B isdesigned for 6-atom repeating-unit backbones, as shown in FIG. 2B. InFIG. 1B, the atom Y linking the 5′ morpholino carbon to the phosphorousgroup may be sulfur, nitrogen, carbon or, preferably, oxygen. The Xmoiety pendant from the phosphorous may be any of the following:fluorine; an alkyl or substituted alkyl; an alkoxy or substitutedalkoxy; a thioalkoxy or substituted thioalkoxy; or, an unsubstituted,monosubstituted, or disubstituted nitrogen, including cyclic structures.Z is sulfur or oxygen, and is preferably oxygen. Particularly preferredmorpholino oligonucleotides include those composed of morpholino subunitstructures of the form shown in FIG. 2B, where X is an amine or alkylamine of the form X═NR₂, where R is independently H or CH₃, that iswhere X═NH₂, X═NHCH₃ or X═N(CH₃)₂, Y═O, and Z═O.

Subunits C-D in FIGS. 1C-D are designed for 7-atom unit-length backbonesas shown for structures in FIGS. 2C and D. In Structure C, the X moietyis as in Structure B, and the moiety Y may be methylene, sulfur, orpreferably oxygen. In Structure D, the X and Y moieties are as inStructure B. In all subunits depicted in FIGS. 1 and 2, each Pi and Pjis a purine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide, and ispreferably selected from adenine, cytosine, guanine and uracil.

As noted above, the substantially uncharged oligomer may advantageouslyinclude a limited number of charged linkages, e.g. up to about 1 perevery 5 uncharged linkages. In the case of the morpholino oligomers,such a charged linkage may be a linkage as represented by any of FIGS.2A-D, preferably FIG. 2B, where X is oxide (—O—) or sulfide (—S—).

More generally, the morpholino oligomers with uncharged backbones areshown in FIGS. 3A-3G. Especially preferred is a substantially unchargedmorpholino oligomer such as illustrated by the phosphorodiamidatemorpholino oligomer (PMO) shown in FIG. 3G. It will be appreciated thata substantially uncharged backbone may include one or more, e.g., up to10-20% of charged intersubunit linkages, typically negatively chargedphosphorous linkages.

Antisense sequence. In the methods of the invention, the antisenseoligomer is designed to hybridize to a region of the target nucleic acidsequence, under physiological conditions with a Tm substantially greaterthan 37° C., e.g., at least 45° C. and preferably 60° C.-80° C., whereinthe target nucleic acid sequence is preferentially expressed inactivated lymphocytes. The oligomer is designed to have high-bindingaffinity to the target nucleic acid sequence and may be 100%complementary thereto, or may include mismatches, e.g., to accommodateallelic variants, as long as the heteroduplex formed between theoligomer and the target nucleic acid sequence is sufficiently stable towithstand the action of cellular nucleases and other modes ofdegradation during its transit from cell to body fluid. Mismatches, ifpresent, are less destabilizing toward the end regions of the hybridduplex than in the middle. The number of mismatches allowed will dependon the length of the oligomer, the percentage of G:C base pair in theduplex and the position of the mismatch(es) in the duplex, according towell understood principles of duplex stability.

Although such an antisense oligomer is not necessarily 100%complementary to a nucleic acid sequence that is preferentiallyexpressed in mature dendritic cells, it is effective to stably andspecifically bind to the target sequence such that expression of thetarget sequence is modulated. The appropriate length of the oligomer toallow stable, effective binding combined with good specificity is about8-40 nucleotide base units, and preferably about 12-25 nucleotides.Oligomer bases that allow degenerate base pairing with target bases arealso contemplated, assuming base-pair specificity with the target ismaintained. mRNA transcribed from the relevant region of a geneassociated with CD86 expression is generally targeted by the antisenseoligonucleotides for use in practicing the invention, however, in somecases double-stranded DNA may be targeted using a non-ionic probedesigned for sequence-specific binding to major-groove sites in duplexDNA. Such probe types are described in U.S. Pat. No. 5,166,315(Summerton et al., 1992), which is hereby incorporated by reference, andare generally referred to herein as antisense oligomers, referring totheir ability to block expression of target genes.

The antisense compound is targeted against an expression-sensitiveregion of a processed or preprocessed CD transcript, that is, a regionwhich, when bound to the antisense compound, is effective to inhibit theexpression of full-length CD86 in dendritic cells. In one generalembodiment, the expression-sensitive region is one that includes or isadjacent the AUG start site of a processed transcript, where formationof an antisense-transcript heteroduplex is effective to inhibittranslation of the transcript. Here the antisense compound has a basesequence that is complementary to a target region containing at least 12contiguous bases in a processed human CD86 transcript, in the targetregion from about −20 to +30 bases with respect to the A nucleotide ofthe AUG start site at position 1, and which includes at least 6contiguous bases of the sequence identified by SEQ ID NO: 9. Exemplaryantisense sequences include those identified as SEQ ID NOS: 21-23, and32.

In a more specific embodiment, the antisense compounds are designed tospan or cover the three bases +12 to +14 bases (where the A nucleotideof the AUG start site represents +1). In this embodiment, the antisensecompound may hybridize to a region spanning these bases, e.g., where thethree bases are in the middle of the target region, or may hybridize toa region predominantly upstream of and including these bases, e.g., thetarget bases extending from −2 to +19 (SEQ ID NO: 23 below), or mayhybridize to a region predominantly downstream of and including thesebases, e.g., the target bases extending from +9 to +30 (SEQ ID NO: 32below).

In another general embodiment, the expression-sensitive region is asplice-site target region that may include (i) an intron regionadjacent, e.g., within 5 bases of, a splice-site donor or acceptorjunction, (ii) a region spanning a donor or acceptor splice-sitejunction, or (iii) the exon region adjacent, e.g., within 5 bases of, asplice-site donor or acceptor junction. The target region preferablycontains at least 12 contiguous bases in a preprocessed human CD86transcript, and includes, in exemplary embodiment, at least 6 contiguousbases of one of the sequences identified by SEQ ID NOS: 10-14. Exemplaryantisense sequences include those identified as SEQ ID NOS: 24-28.

However, in some cases, other regions of the CD86 mRNA (SEQ ID NO: 29)may be targeted, including one or more of, an initiator or promotersite, a 3′-untranslated region, and a 5′-untranslated region. Bothspliced and unspliced, preprocessed RNA may serve as the template fordesign of antisense oligomers for use in the methods of the invention.

When the antisense compound is complementary to a specific region of atarget gene (such as the region adjacent the AUG start codon of the CD86gene) the method can be used to monitor the binding of the oligomer tothe CD86 RNA.

The antisense compounds for use in practicing the invention can besynthesized by stepwise solid-phase synthesis, employing methodsdetailed in the references cited above. The sequence of subunitadditions will be determined by the selected base sequence. In somecases, it may be desirable to add additional chemical moieties to theoligomer compounds, e.g. to enhance the pharmacokinetics of the compoundor to facilitate capture or detection of the compound. Such a moiety maybe covalently attached, typically to the 5′- or 3′-end of the oligomer,according to standard synthesis methods. For example, addition of apolyethyleneglycol moiety or other hydrophilic polymer, e.g., one having10-100 polymer subunits, may be useful in enhancing solubility. One ormore charged groups, e.g., anionic charged groups such as an organicacid, may enhance cell uptake. A reporter moiety, such as fluorescein ora radiolabeled group, may be attached for purposes of detection.

Alternatively, the reporter label attached to the oligomer may be aligand, such as an antigen or biotin, capable of binding a labeledantibody or streptavidin. In selecting a moiety for attachment ormodification of an oligomer antisense, it is generally of coursedesirable to select chemical compounds of groups that are biocompatibleand likely to be tolerated by cells in vitro or in vivo withoutundesirable side effects.

B. Arginine-Rich Polypeptide Moiety

The use of arginine-rich peptide sequences conjugated to unchargedantisense compounds, e.g., PMO, has been shown to enhance cellularuptake in a variety of cells (Wender, Mitchell et al. 2000; Moulton,Hase et al. 2003; Moulton and Moulton 2003) (Iversen, Moulton et al.U.S. Patent Application No. 60/466,703, now U.S. publication No.2004/0265879 A1, published Dec. 30, 2004, all of which are incorporatedherein by reference.

In one embodiment of the invention, the antisense compound is covalentlylinked at its 3′ or 5′ end to an arginine rich-peptide effective toenhance uptake of the compound into dendritic cells relative to uptakein the absence of the peptide. The arginine-rich peptide is detailed inthe above references to Moulton et al., and described in U.S. patentapplication. Preferably, the peptide is composed of d-amino acids,1-amino acids, non-natural amino acids or a combination thereof.Exemplary arginine-rich peptides include those identified by SEQ ID NOS:1-3, of which those identified as SEQ ID NOS: 1 and 2 are preferred.

The transport peptide may significantly enhance cell entry of attacheduncharged oligomer compounds, relative to uptake of the compound in theabsence of the attached transport moiety, and relative to uptake by anattached transport moiety lacking the hydrophobic subunits Y. Suchenhanced uptake is preferably evidenced by at least a two-fold increase,and preferably a four-fold increase, in the uptake of the compound intomammalian cells relative to uptake of the agent by an attached transportmoiety lacking the hydrophobic subunits Y. Uptake is preferably enhancedat least twenty fold, and more preferably forty fold, relative to theunconjugated compound.

A further benefit of the transport moiety is its expected ability tostabilize a duplex between an antisense oligomer and its target nucleicacid sequence, presumably by virtue of electrostatic interaction betweenthe positively charged transport moiety and the negatively chargednucleic acid. The number of charged subunits in the transporter is lessthan 14, as noted above, and preferably between 8 and 11, since too higha number of charged subunits may lead to a reduction in sequencespecificity.

The transport moiety may also lower the effective concentration of anantisense oligomer to achieve antisense activity as measured in bothtissue culture and cell-free systems. Cell-free translation systemsprovide an independent means to assess the enhanced effect of thetransport moiety on the antisense oligomer's ability to bind to itstarget and, through steric blocking, inhibit translation of downstreamsequences.

C. rTAT (P002) Peptide

In studies conducted in support of the present invention, severaldifferent “arginine-rich” peptide sequences were conjugated tofluorescent tagged PMO (PMO-fl) and examined to determine the effect ofpeptide sequence on uptake into lymphocytes Enhanced uptake was observedfor all arginine-rich peptide-PMO conjugates tested compared tounconjugated PMO. The P003 and P005 arginine-rich peptides (SEQ ID NOS:2 and 3, respectively) provide enhanced uptake into lymphocytesregardless of the cell activation state. However, among thearginine-rich peptides examined, the rTAT (P002) peptide[NH₂—RRRQRRKKRC—COOH] (SEQ ID NO: 1) PMO conjugate exhibiteddifferential uptake into dendritic cells dependent on cell activationstatus. PMO uptake was greatly increased in mature dendritic cells asshown below as well as activated B cells and CD4 and CD8 T cells whencompared to naïve lymphocytes (Mourich, Moulton et al. U.S. PatentApplication No. 60/505,418). Furthermore, experiments in support of theinvention demonstrate that the arginine-rich peptide-antisense compoundsconjugates alone do not affect the maturation state of dendritic cells.This was shown by treating dendritic cells with arginine-richpeptide-PMO conjugates in the absence of a maturation stimulus andobserving no activation of the dendritic cell population.

The rTAT peptide can be synthesized by a variety of known methods,including solid-phase synthesis. The amino acid subunits used inconstruction of the polypeptide may be either I- or d-amino acids,preferably all I-amino acids or all d-amino acids. Minor (or neutral)amino acid substitutions are allowed, as long as these do notsubstantially degrade the ability of the polypeptide to enhance uptakeof antisense compounds selectively into activated T cells. One skilledin the art can readily determine the effect of amino acid substitutionsby construction of a series of substituted rTAT polypeptides, e.g., witha given amino acid substitution separately at each of the positionsalong the rTAT chain. Using the above test for uptake of fluoresceinatedPMO-polypeptide conjugate, one can then determine which substitutionsare neutral and which significantly degrade the transporter activity ofthe peptide. Rules for neutral amino acid substitutions, based on commoncharge and hydrophobicity similarities among distinct classes of aminoacids are well known and applicable here. In addition, it will berecognized that the C-terminal cysteine of SEQ ID NO: 1 is added forpurposes of coupling to the antisense compound, and may bereplaced/deleted when another terminal amino acid or linker is used forcoupling.

The rTAT polypeptide can be linked to the compound to be delivered by avariety of methods available to one of skill in the art. The linkagepoint can be at various locations along the transporter. In selectedembodiments, it is at a terminus of the transporter, e.g., theC-terminal or N-terminal amino acid. In one exemplary approach, thepolypeptide has, as its C-terminal residue, a single cysteine residuewhose side chain thiol is used for linking. Multiple transporters can beattached to a single compound if desired.

When the compound is a PMO, the transporter can be attached at the 5′end of the PMO, e.g. via the 5′-hydroxyl group, or via an amine cappingmoiety, as described (Moulton and Moulton 2003) (Iversen, Moulton et al.U.S. Patent Application No. 60/466,703). Alternatively, the transportermay be attached at the 3′ end, e.g. via a morpholino ring nitrogen, asdescribed (Moulton and Moulton 2003) (Iversen, Moulton et al. U.S.Patent Application No. 60/466,703), either at a terminus or an internallinkage. The linker may also comprise a direct bond between the carboxyterminus of a transporter peptide and an amine or hydroxy group of thePMO, formed by condensation promoted by, for example carbodiimide.

Linkers can be selected from those which are non-cleavable under normalconditions of use, e.g., containing a thioether or carbamate bond. Insome embodiments, it may be desirable to include a linkage between thetransporter moiety and compound which is cleavable in vivo. Bonds whichare cleavable in vivo are known in the art and include, for example,carboxylic acid esters, which are hydrolyzed enzymatically, anddisulfides, which are cleaved in the presence of glutathione. It mayalso be feasible to cleave a photolytically cleavable linkage, such asan ortho-nitrophenyl ether, in vivo by application of radiation of theappropriate wavelength.

For example, the preparation of a conjugate having a disulfide linker,using the reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP)or succinimidyloxycarbonyl α-methyl-α-(2-pyridyldithio) toluene (SMPT),is described (Moulton and Moulton 2003) (Iversen, Moulton et al. U.S.Patent Application No. 60/466,703). Exemplary heterobifunctional linkingagents which further contain a cleavable disulfide group includeN-hydroxysuccinimidyl 3-[(4-azidophenyl)dithio] propionate and others.

IV. Selective Uptake of rTAT-Antisense Oligomers into ActivatedDendritic Cells

The present invention provides a method and composition for deliveringtherapeutic compounds, e.g., uncharged antisense compounds, specificallyto activated immune cells, e.g., antigen-activated T cells, B cells, andmature dendritic cells.

The ability of the rTat (SEQ ID NO:1, P002) peptide to enhance uptake ofa fluoresceinated PMO antisense compound selectively into activatedmouse dendritic cells is demonstrated in the study described in Example1, and with the results shown in FIG. 5. In this study, cultured mousedendritic cells were incubated with fluorescein-labeled P002-PMOconjugate and subjected to lymphocyte activating substances, asdescribed in Example 1. Dendritic cells were stained with antibody todetermine the extent of uptake by FACS analysis of the cells. Theresults show relatively low uptake of the antisense PMO intonon-activated dendritic cells. Activation by lipopolysaccharide (LPS)caused significantly increased uptake of the antisense oligomer intodendritic cells.

The property of activation-dependent uptake of peptide-antisenseconjugate is not observed with other arginine-rich peptides, which areknown to enhance drug transport into cells. This is also demonstrated bythe study described in Example 1, and with the results shown in FIG. 5.As seen in these figures, P003-PMO conjugate (corresponding to thearginine-rich peptide of SEQ ID NO: 2) is readily taken up by immaturedendritic cells. PMO alone is relatively poorly taken up by immaturedendritic cells, and P002-PMO shows enhanced uptake into LPS treateddendritic cells.

In one aspect of the invention, therefore, the P002 peptide may beconjugated to a substantially uncharged antisense compound, to enhanceits uptake selectively into antigen-activated, mature dendritic cells,including antigen-activated, mature human dendritic cells.

V. Treating Transplantation Rejection and Autoimmune Disorder

By manipulating the immune system's normal mechanism for the generationof immune tolerance to self antigens, the present invention provides amethod and composition that alters the function and activity of maturedendritic cells in a way that is advantageous in the treatment oftransplantation rejection or autoimmune disorders, such as multiplesclerosis, lupis, myathenia gravis, inflammatory bowel disease andrheumatoid arthritis.

By employing an antisense compound against CD86 (e.g., SEQ ID NOS: 21-28and 32), the present invention provides a means to precisely andspecifically block T cell activation to an antigen presented by a maturedendritic cell. This allows the generation of a tolerized T cell anddendritic cell population responding to transplanted tissue, whenchronically activated as in an autoimmune condition, or by animmunogenic therapeutic protein. Where the antisense compound is linkedto an rTAT peptide, the conjugate preferentially targets activateddendritic cells, thus allowing the therapy to be made highly specificfor mature dendritic cells.

The generation of tolerized, anergic T-cells using the compounds andmethods of the invention also provides a long-lasting tolerance that hasa variety of therapeutic advantages.

A. CD86 Antisense Oligomers, T Cell Costimulation and IL10 Production byMature Dendritic Cells.

Dendritic cells (DCs) reside and traffic through most tissues of thebody in an immature state. Upon encountering an inflammatory stimuluschanges in DC phenotype rapidly ensue. Hallmarks of this phenotypicshift termed “maturation” include the loss of phagocytic function,increased surface expression of MHC class I, II, adhesion molecules,distinct chemokine receptors and costimulatory molecules such as B7-1(CD80) and B7-2 (CD86). Together these provide mature DCs the ability totraffic to lymphoid tissue and the capacity to be potent antigenpresenting cells (APCs) to naïve T cells. The cascade of signalingevents that follow when CD28 on the responding T cell is engaged by CD86are well established. However, little is known about the reciprocity ofevents occurring in APCs due to the expression or engagement of CD80 andCD86 molecules. Studies to determine if antisense molecules could enterDCs and be used to inhibit the expression of CD86 molecules ledunexpectedly to the observations described in the present invention thatAPCs, specifically dendritic cells, undergo important alterations whenB7 molecules are engaged. These observations include a link between theexpression of CD86 and the regulation of IL-10 expression in bonemarrow-derived mature DCs.

Exemplary target sequences for the CD86 (B7-2) gene are listed in Table1 below. The murine CD86 sequences are noted with “mu” and are derivedfrom Genbank Accession No. AF065898. The human CD86 AUG target andtargeting sequences are noted with “hu” and derived from GenbankAccession No. NM006889. The human Exon 6, 7, and 8 splice donor (sd) andsplice acceptor (sa) target and targeting sequences are derived fromGenbank Accession Nos. U17720, U17721 and U17722, respectively. TABLE 1Exemplary CD86 Target Sequences Oligomer SED ID Target Sequence (5′ to3′) Sp. Nct. Range NO. CD86 AUG cggaagcacccacgatggaccccag mu 19-43 4Exon 7sa gctgtttccgtggagacgc mu  99-117 5 Exon 9sd gccgaatcagcttagcaggmu 833-851 6 Exon 10sa gcccagcaacacagcctct mu 851-869 7 Exon 11sagaaaccaaatgcagagtg mu 944-961 8 CD86 AUGcatttgtgacagcactatgggactgagtaacattct hu 132-177 9 ctttgtgatg CD86Ex6saagcttgaggaccctcagcctc hu 170-190 10 CD86Ex6sd gcctcgcaactcttataaatgtg hu291-313 11 CD86Ex7sa gaaccaacacaatggagaggga hu 274-295 12 CD86Ex7sdgagtgaacagaccaagaaaag hu 298-319 13 CD86Ex8sa agaaaaaatccatatacctgaa hu223-244 14

TABLE 2 Exemplary CD86 Targeting Sequences Oligomer SEQ ID TargetSequence (5′ to 3′) Sp. NO. B7-2 AUG1 CTGGGGTCCATCGTGGGTGC mu 15 B7-2AUG2 GGGGTCCATCGTGGGTGCTTCCG mu 16 Exon 7sa GCGTCTCCACGGAAACAGC mu 17Exon 9sd CCTGCTAAGCTGATTCGGC mu 18 Exon 10sa AGAGGCTGTGTTGCTGGGC mu 19Exon 11sa CACTCTGCATTTGGTTTC mu 20 CD86 AUG1 GTTACTCAGTCCCATAGTGCTG hu21 CD86 AUG2 CCATAGTGCTGTCACAAATG hu 22 CD86 AUG3 GAATGTTACTCAGTCCCATAGhu 23 CD86Ex6sa GAGGCTGAGGGTCCTCAAGCT hu 24 CD86Ex6sdCACATTTATAAGAGTTGCGAGGC hu 25 CD86Ex7sa TCCCTCTCCATTGTGTTGGTTC hu 26CD86Ex7sd CTTTTCTTGGTCTGTTCACTC hu 27 CD86Ex8sa TTCAGGTATATGGATTTTTTCThu 28 CD86 AUG4 CATCACAAAGAGAATGTTACTC hu 32

B. Treatment Methods

In one aspect, the invention is directed to methods of inducingimmunological tolerance in vivo in a patient, by administering to thepatient a therapeutically effective amount of a peptide-conjugated CD86PMO pharmaceutical composition, as described herein, e.g., apharmaceutical composition comprising an antisense oligomer to CD86.

The antisense oligomers of the invention can be effective in thetreatment of patients by modulating the immunological response toallogeneic transplantation or elimination of chronically activated Tcells in the case of autoimmune diseases.

In one embodiment, a subject is in need of tolerized dendritic cells andT cells when responding to an allogeneic transplantation. In thisembodiment, a CD86 antisense compound is administered to the subject ina manner effective to result in blocking the formation of activated Tcells. Typically, the patient is treated with the conjugate shortlybefore, e.g., a few days before, receiving the transplant, then treatedperiodically, e.g., once every 14 days, until immunological tolerance isestablished. Immunological tolerance can be monitored during treatmentby testing patient T cells for reactivity with donor MHC antigens in astandard in vitro test, as detailed below.

For the treatment of an autoimmune disorder, such as multiple sclerosis,lupis, myathenia gravis, inflammatory bowel disease and rheumatoidarthritis, the patient is given an initial single dose of the CD86antisense conjugate, then additional doses on a periodic basis, e.g.,every 3-14 days, until improvement in the disorder is observed. Asabove, development of immunological tolerance can be monitored duringtreatment by testing T cells from a blood sample for their ability toreact with a selected, relevant antigen in vitro.

It will be understood that in vivo administration of such a CD86antisense compound is dependent upon, (1) the duration, dose andfrequency of antisense administration, and (2) the general condition ofthe subject. A suitable dose can be approximated from animal modelstudies and extrapolated to patient weight.

Typically, one or more doses of CD86 antisense oligomer areadministered, generally at regular intervals for a period of about oneto two weeks. Preferred doses for oral administration are from about 1mg oligomer/patient to about 25 mg oligomer/patient (based on an adultweight of 70 kg). In some cases, doses of greater than 25 mgoligomer/patient may be necessary. For IV administration, the preferreddoses are from about 1.0 mg oligomer/patient to about 100 mgoligomer/patient, preferably 5-50 mg oligomer/patient, (based on anadult weight of 70 kg). The antisense agent is generally administered inan amount sufficient to result in a peak blood concentration of at least200-400 nM antisense oligomer.

In general, the method comprises administering to a subject, in asuitable pharmaceutical carrier, an amount of a CD86 antisense oligomer,e.g., morpholino oligomer, effective to inhibit expression of CD86 andincrease expression of IL-10 in dendritic cells.

Effective delivery of an antisense oligomer to the target nucleic acidis an important aspect of the methods described herein. In accordancewith the invention, such routes of antisense oligomer delivery include,but are not limited to, inhalation; transdermal delivery; varioussystemic routes, including oral and parenteral routes, e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular delivery.

It is appreciated that any methods which are effective to deliver a CD86PMO to the cells of an allogeneic transplant or to introduce the agentinto the bloodstream are also contemplated.

In preferred applications of the method, the subject is a human subjectand the methods of the invention are applicable to treatment of anycondition wherein promoting immunological tolerance would be effectiveto result in an improved therapeutic outcome for the subject undertreatment.

It will be understood that an effective in vivo treatment regimen usinga CD86 antisense compound in the methods of the invention will varyaccording to the frequency and route of administration as well as thecondition of the subject under treatment. Accordingly, such in vivotherapy will generally require monitoring by tests appropriate to thecondition being treated and a corresponding adjustment in the dose ortreatment regimen in order to achieve an optimal therapeutic outcome.

C. Ex Vivo Treatment of Human Dendritic Cells

In another preferred application of the method, autologous dendriticcells isolated from a human subject can be treated ex vivo with the CD86antisense compound in the presence of a selected, relevant antigen.Studies in several systems have demonstrated that when dendritic cellsare pulsed with antigens ex vivo, and these cells are subsequentlyreadministered to the human subject from whom they were isolated,specific immunity can be established (Lu, Arraes et al. 2004;Mohamadzadeh and Luftig 2004). A similar strategy can be used toestablish, ex vivo, a tolerogenic population of dendritic cells usingthe methods and compositions of the present invention. Dendritic cellsare isolated from the peripheral blood of a human subject using methodswell-known to those skilled in the art. Growth and treatment of thedendritic cells with the relevant antigen and antisense CD86 antisensewill induce the formation of dendritic cells that, upon readministrationto the subject, will condition the dendritic cells to induce a T-cellresponse that suppresses the antigen-specific immunity. This applicationof the method is particularly useful in treating an autoimmune disorderwhere the immune system is reacting inappropriately to specific antigensand these antigens can be used to condition the dendritic cells. Anexample is the immune-mediated destruction of myelin in multiplesclerosis (MS). Myelin basic protein (MBP) and proteolipid protein (PLP)are host proteins which are thought to be the key antigens in theetiology of this autoimmune disease (Shevac 2002).

D. Administration of Anti-CD86 Antisense Oligomers

Transdermal delivery of an antisense oligomer may be accomplished by useof a pharmaceutically acceptable carrier. One example of morpholinooligomer delivery is described in PCT patent application WO 97/40854,incorporated herein by reference.

In one preferred embodiment, the oligomer is an anti-CD86 morpholinooligomer, contained in a pharmaceutically acceptable carrier, anddelivered orally. In a further aspect of this embodiment, the antisenseoligomer is administered at regular intervals for a short time period,e.g., daily for two weeks or less. However, in some cases the antisenseoligomer is administered intermittently over a longer period of time.

It follows that a morpholino antisense oligonucleotide composition maybe administered in any convenient vehicle, which is physiologicallyacceptable. Such an oligonucleotide composition may include any of avariety of standard pharmaceutically accepted carriers employed by thoseof ordinary skill in the art. Examples of such pharmaceutical carriersinclude, but are not limited to, saline, phosphate buffered saline(PBS), water, aqueous ethanol, emulsions such as oil/water emulsions,triglyceride emulsions, wetting agents, tablets and capsules. It will beunderstood that the choice of suitable physiologically acceptablecarrier will vary dependent upon the chosen mode of administration.

In some instances liposomes may be employed to facilitate uptake of anantisense oligonucleotide into cells. (See, e.g., Williams, 1996;Lappalainen, et al., 1994; Uhlmann, et al., 1990; Gregoriadis, 1979.)Hydrogels may also be used as vehicles for antisense oligomeradministration, for example, as described in WO 93/01286. Alternatively,an oligonucleotide may be administered in microspheres ormicroparticles. (See, e.g., Wu et al., 1987).

Sustained release compositions are also contemplated within the scope ofthis application. These may include semipermeable polymeric matrices inthe form of shaped articles such as films or microcapsules.

E. Monitoring Treatment

The efficacy of a given therapeutic regimen involving the methodsdescribed herein, may be monitored, e.g., by conventional FACS assaysfor the phenotype of cells in the circulation of the subject undertreatment or cells in culture. Such analysis is useful to monitorchanges in the numbers of cells of various lineages, in particular,activated T and B cells in response to an allogeneic transplant.

Phenotypic analysis is generally carried out using monoclonal antibodiesspecific to the cell type being analyzed. The use of monoclonalantibodies in such phenotypic analyses is routinely employed by those ofskill in the art for cellular analyses and monoclonal antibodiesspecific to particular cell types are commercially available.

The CD86 PMO treatment regimen may be adjusted (dose, frequency, route,etc.), as indicated, based on the results of the phenotypic andbiological assays described above.

From the foregoing, it will be appreciated how various objects andfeatures of the invention are met. The specific blockade of dendriticcell activation of T cells capable of rejecting transplanted tissues isan important therapy for numerous human diseases where immunologicaltolerance is beneficial. The present invention provides a method ofspecifically blocking the activation of these cells through the use ofantisense oligomers designed to inhibit CD86 expression, or specificportions of CD86, during the stage of antigen-specific activation andthe generation of anergic, tolerized T cells. Antisense CD86 mediatedsuppression of either chronically activated T cells (i.e. autoimmunity)or naïve T cell responding to alloantigens (transplantation) provides apotent and specific therapeutic effect.

Additionally, this treatment method is long lived because the immunesystem is unable to replenish antigen-specific T cell clones once theprecursor population is removed from the T cell repertoire. In addition,by specifically targeting the antisense CD86 oligomer to maturedendritic cells, immature dendritic cells would be unaffected, allowingfor the patient to respond normally to foreign antigens as soon as thetherapy is withdrawn. Moreover, the immune status of the patient priorto the antisense CD86 therapy (e.g. immunity provided by previousvaccinations or infections) would remain intact.

The following examples illustrate but are not intended in any way tolimit the invention.

Materials and Methods

A. Antisense Oligomers and Peptide Conjugates

PMO synthesis, peptide conjugation and purification were preformed atAVI BioPharma Inc. (Corvallis, Oreg.) as previously described (Summertonand Weller 1997). Oligomer sequences were designed to either blocktranslation by binding to bases surrounding the AUG start site (CAT) oralter RNA splicing by blocking splice donor or splice acceptor sites, saor sd, respectively. An oligomer with the same base composition of theB7-2 (AUG) in a scrambled order was synthesized to serve as a controlfor oligomer treatment. B7-1 and B7-2 antisense oligomer sequences weredesigned using Genbank sequences accession numbers X60958 and U39459-66,respectively, The PMO sequences and designations are as follows; B7-1(AUG) 5′-GCA AGC CAT AGC TTC AGA TGC-3′ (SEQ ID NO:29), B7-2(AUG) 5′-CTGGG GTC CAT CGT GGG TGC-3′(SEQ ID NO: 15), EXON 7sa 5′-GCG TCT CCA CGGAAA CAG C-3′ (SEQ ID NO:17), EXON 9sd 5′-CCT GCT MG CTG ATT CGG C-3′(SEQ ID NO:18), EXON 10sa 5′-AGA GGC TGT GTT GCT GGG C-3′ (SEQ IDNO:19), EXON 11 sa 5′-CAC TCT GCA TTT GGT TTC-3′ (SEQ ID NO:20),SCRAMBLE 5′-CGT GGT GCA CTG CGT GTG GC-3′ (SEQ ID NO:30), 705-FL 5′-CCTCTT ACC TCA GTT ACA-FL-3′ (SEQ ID NO:31). The 3′ fluorescein conjugatedoligomer 705-FL targets an irrelevant gene (human b-globin intron 2) andwas used to analyze the intracellular delivery properties of differentpeptides into cultured DCs. Three different arginine rich peptidesequences were conjugated separately to the PMOs used in this study;P002=N-RRRQRRKKRGYC-CONH₂ (SEQ ID NO:1), P003=N-RRRRRRRRRFFC-CONH₂(SEQID NO:2) and P005=N-RRRQRRKKRGYFFC-CONH₂ (SEQ ID NO:3).

B. Generation and Culturing of Bone Marrow Derived DCs

Murine DCs were generated by culturing marrow flushed from the femur andtibia of female BALB/c mice obtained from Jackson Laboratory aged 6-12weeks. The marrow was minced through a 70 micron nylon cell strainer (BDFalcon) and washed twice after centrifugation in DMEM+1% fetal bovineserum (FBS) and penicillin streptomycin and glutamine (PSG). Cellsuspension were made in culture medium (RPMI+10% FBS, PSG and 5×10⁻⁵ M2-mecapotoethanol) supplemented with recombinant mouse GM-CSF(eBioscience) [25 ng/ml] and seeded onto a 100 mm bacteriological Petridish (Falcon) at 2×10⁵ cells/ml. After 3 days an additional 5 ml offresh medium was added containing GM-CSF. The culture supernatant wasremoved on the 6^(th) day and centrifuged to recover any dislodgedcells. The cell pellet was suspended in 10 ml fresh media with GM-CSF,placed back on to the original 100 mm dish and cultured for anadditional 2-4 days prior to treatment with PMO.

Non-adherent cells were harvested by gentle pipetting of the mediumwhich was then transferred to a tube for centrifugation at roomtemperature. The cell pellet was washed twice and then enumerated. Thewells of a 12 well plate were seeded with 1.5 ml [5×10⁵ cells/ml] infresh culture medium containing GM-CSF. PMO working stock [1 mM] insterile water was added directly to the wells to obtain a finalconcentration ranging from 2-20 μM 2-4 hours prior to inducingmaturation. Control culture wells were treated with the equivalentamount of sterile water. Maturation was induced by the addition oflipopolysaccharide (LPS) E. coli 026:B6 (Sigma) [1 μg/ml] or anti-CD40(eBioscience) at [5 μg/ml] for 16 hours following PMO treatment. Cultureconditions to block binding of cell associated ligand to B7 moleculeswas carried out by addition of [5 μg/ml] recombinant chimeric CTLA-4 FCnon-cytolytic molecule (Chimerigen Allston, Mass.). Cultures used foranalysis of IL-4 and IL-12 cytokine production were treated with 1 μl ofthe protein transport inhibitor brefeldin A GolgiPlug (BD Pharmingen)for the last 4 hrs of incubation.

C. Flow Cytometric Analysis

Cells were removed from culture wells by scrapping with a 25 cm cellscraper (Sarstedt) and rinsing with 1 ml cold FACS buffer [PBS+2%FBS+0.2% sodium azide]. The cells were washed twice in cold FACS bufferafter centrifugation and suspended in 50 ml FACS buffer containing 1 μganti-mouse CD16/CD32 FC blocking antibody (eBioscience) for 15 min onice. The FC blocked samples were centrifuged and suspended in 50 mlantibody staining reagent for 30 min on ice. The surface stainingreagents used were; CD111c-APC (BD Bioscience) and CD86-PE, CD86-FITCand CD80-PE (eBioscience) diluted in 50 ml cold FACS buffer. Stains werecombined in the 50 ml when dual surface staining was needed foranalysis. Cells were washed thrice by centrifugation in FACS bufferprior to analysis or use in additional staining procedures.

Intracellular cytokine staining was performed immediately followingsurface staining and washes. The cells were fixed and permeabilized in100 μl Cytofix/Cytoperm (Pharmingen) buffer for 20 min on ice. Cellpellets were suspended in 50 μl of 1× PermNVash buffer (Pharmingen)containing either IL-10-FITC or IL-12-APC and IL-4-FITC (Pharmingen) andincubated for 30 min on ice. The cells were washed thrice and suspendedin 300 ml FACS buffer prior collection of flow cytometric data on a FACScaliber cytometer (Becton Dickinson). Cytometric data was analyzed usingFCS Express Software (Denovo Software).

D. RT-PCR

After treatment with PMO conjugates for 4 hours and then for 16 hourswith LPS [1.0 μg/ml] total cellular RNA was isolated from the culturedcells using RNAeasy Mini kit (Qiagen) according to manufacture'sinstructions. The isolated RNA was treated with RNAse free DNAse I (2 U)for 30 min at 37° C. to eliminate contaminating genomic DNA followed byheating to 70° C. for 20 min to inactivation the enzyme. This materialwas used as template for single-tube reverse transcription andpolymerase chain reaction using SuperScript One-Step RT-PCR withPlatinum Taq enzyme (Invitrogen). Primers spanning 870 bp of the B7-2mRNA, forward primer 5′-GGCAATCCTTATCTTTGTGACAGTC-3′ and reverse primer:5′-TTTGCTGMGCMTTTGGGG-3′ were used to examine splice altering activityof the PMOs. Primers to detect mouse IL-10 mRNA forward primer5′-GATCCAGGGATCTTAGCTMCGG-3′ and reverse primer5′-TTCTCTTCCCMGACCCATGAGT-3′ spanning 406 bp were derived from theGenbank sequence accession number NM_(—)010548 bases 675-1081.

The resulting amplicons were fractionated on an EtBr stained 3.0%agarose gel to determine size. Altered splicing patterns and continuityof the open reading frames were confirmed by sequencing after insertioninto plasmid vector using the TOPO TA cloning kit according tomanufacture's instructions (Invitrogen). At least three clones harboringthe different amplicons were examined. Identity and sequence alignmentswere performed by BLAST search analysis.

EXAMPLE 1 Arginine-Rich Peptides Enhance Uptake of Oligomers into MatureDendritic Cells

Delivery of antisense molecules without substantial manipulation of thecellular membrane has been an impediment to targeting gene expression inDCs and other immune cell types. These procedures often result inextensive damage to the membrane allowing for only short livedexperiments to be conducted. Numerous arginine-rich peptides wereexamined as to their ability to deliver oligomers to various cell typeswith no manipulation beyond direct addition to cells cultured undernormal conditions. The PMO chemical structure and peptides used in thisstudy are shown in FIG. 4. PMO synthesis and conjugation of peptides andor fluorescein were carried out at AVI BioPharma as previously described(Summerton and Weller 1997; Moulton, Hase et al. 2003; Moulton, Nelsonet al. 2004). The arginine-rich peptides shown in FIG. 4 are amongseveral that have been shown to enhance cellular uptake. Fluorescein canbe linked to the 3′ end of the peptide-PMO conjugate to allow imagingand or detection of PMO uptake in intact cells.

Using a fluorescein linked PMO, FIG. 5 shows that bone marrow derivedDCs readily take up PMO conjugates of the P003 peptide (FIG. 5, SEQ IDNO:2). In the experiment represented in this figure an irrelevantcontrol PMO (705, 5′-CCTCTTACCTCAGTTACA-3′) was used to measure uptakeof unconjugated and peptide-conjugated PMOs into DCs. Peptides withsimilar amino acid content but varied sequence such as P005 (SEQ IDNO:3) perform similarly. Surprisingly, when PMO conjugates of P002peptide (SEQ ID NO:1) were tested it was observed that uptake into DCswas enhanced after stimulation with lipopolysaccharide (LPS) compared tountreated or immature DCs (FIG. 5).

The data presented in FIG. 5 were obtained using murine DCs obtainedfrom murine bone marrow cells cultured for 8 days in RPMI+10% FBSsupplemented with granulocyte macrophage colony stimulating factor(GM-CSF) (25 ng/ml) and treated in duplicate wells with either naked orpeptide conjugated PMO 705 [5 mM] linked to fluorescein. One well foreach oligomer treatment received LPS [1 mg/ml]. The cells were culturedfor 16 hrs and then harvested, washed 3 times with PBS, stained withCD11 c-APC and analyzed by flow cytometry. The histogram indicates thelevel of fluorescein in the CD11c positive (i.e. mature DC) cellpopulation.

EXAMPLE 2 Antisense Inhibition of CD86 Expression Also Alters CD80Expression

To determine if the enhanced uptake of the PMO into DCs provided by thepeptide conjugates would translate into functional antisense activity wechose to synthesize oligomers targeting the translational start site ofCD86 (B7-2 AUG1, SEQ ID NO:15) and a sequence scrambled control oligomer(5′-CGTGGTGCACTGCGTGTGGC-3′). A considerable reduction in the level ofCD86 (B7-2) was observed in cultured DCs after treatment with thesequence-specific oligomer and LPS compared to controls (FIG. 6, tophistogram). However, when a measure of the level of CD80 (B7-1) was madeunder the same conditions it was observed that a significant reductionwas produced in the cultures treated with antisense to CD86 compared tocontrols (FIG. 6, bottom histogram). This was unexpected since the CD86sequence shares little homology with that of CD80 and considerably lowlevels of homology around the translational start site. Nearly identicalresults were observed with regards to a reduction in CD80 when anoligomer targeting a different sequence (B7-2 AUG2, SEQ ID NO:16)surrounding the CD86 translational start site was used (data not shown).

The data in FIG. 6 were generated using bone marrow derived DCs treatedin duplicate with either P002 peptide conjugated PMO [20 mM] antisenseto CD86 (SEQ ID NO: 15), scrambled PMO sequence(5′-CGTGGTGCACTGCGTGTGGC-3′, SEQ ID NO:30) or media alone for 4 hours.LPS [1.0 mg/ml] was then added to all cultures for 16 hours. The cellswere washed, stained with CD11c-APC antibody (i.e. specific for matureDCs) and either anti-mouse CD80-PE or CD86-PE antibodies and analyzed byflow cytometry.

EXAMPLE 3 Increased DC IL-10 Production is Linked to Diminished CD86Expression and Not Ligand Interaction

In light of the results seen in Example 2 we examined the cytokineproduction profile of the CD86 antisense treated DCs. IL-4 and IL-12production was not significantly altered in the DCs receiving the B7-2AUG1 CD86 antisense oligomer (SEQ ID NO:15) compared to controls (datanot shown). However, IL-10 production was evident when DCs were treatedwith a maturation stimulus such as LPS in conjunction with the CD86antisense oligomer-P002 conjugate (SEQ ID NO:15) and not the P002peptide alone or a scrambled control sequence conjugated to P002 asshown in Table 3 below. The same result was seen when other deliverypeptides were used with SEQ ID NO:15 or alternate sequences targetingthe translational start site (SEQ ID NO:16). Furthermore, IL-10 stainingwas detectable when the DCs were not permeabilized indicating that IL-10was being secreted from the cells where it could exert an autocrine orparacrine effect (data not shown). TABLE 3 Inhibition of CD86 InducesIL-10 Production in LPS-treated Dendritic Cells % DCs % DCs TreatmentCD86 Positive IL10 Positive Control (No LPS) 6.92 0.10 P002 Peptide (NoLPS) 6.47 0.10 Scramble-P002 + LPS 13.4 0.75 B7-2 AUG1(SEQ ID NO: 15)-2.26 21.1 P002(SEQ ID NO: 1) + LPS

The data presented in Table 3 was generated using murine bone marrowderived DCs treated in duplicate with either P002 peptide (SEQ ID NO:1)alone, P002 conjugated to PMO antisense CD86 (SEQ ID NO:15) or scrambledPMO sequence (5′-CGTGGTGCACTGCGTGTGGC-3′, SEQ ID NO:30) at 20 mM ormedia alone for 4 hours. LPS [1.0 mg/ml] was then added to theappropriate cultures for 16 hours. The cell were washed, FC blocked,then stained with antibody specific for mature DCs (CD11c-APC) andantibody specific for CD86 (CD86-PE). Intracellular staining withanti-IL-10-FITC antibody was carried out after fixation andpermeabilization of the cells. The numbers indicate the percentage ofCD11c positive cells (mature DCs) staining positive for IL-10 and CD86respectively.

We also examined whether the regulation of IL-10 in the mature DCs wasdue to the loss of some interaction with a yet unknown ligand that ispresent in bone marrow derived DCs cultures or whether it might be dueto the loss of control through the absences of CD86 (B7-2). Arecombinant form of the CTLA-4 molecule, a receptor on T cells for CD86,was used to block interactions of the CD86 molecule in cultured DCs. DCsreceiving this treatment were compared to CD86 antisense-treated DCs asto the levels of IL-10 produced under two different conditions of DCsmaturation. DCs received a maturation stimulus from either LPS oranti-CD40 and were then stained for intracellular IL-10. Under eithermaturation condition IL-10 was only significantly produced in theP002-conjugated CD86 antisense (SEQ ID NO: 15) treated cells compared tocontrol untreated cells (FIG. 7). This suggests that the regulation ofIL-10 production in maturing DCs is linked to levels of CD86 expressionand not those of CD86 interactions with a ligand.

The data in FIG. 7, which shows blocking CD86 interactions does not leadto IL-10 expression in bone marrow derived DCs, were generated bytreatment of murine bone marrow DCs in duplicate with either mediaalone, antisense CD86-P003 conjugate [2 mM] or CTLA-4 Ig [5.0 mg/ml] for4 hours. One of the duplicate cultures was treated with LPS [1.0 mg/ml]and the other with anti-CD40 [5.0 mg/ml] for 16 hours to inducematuration. The cells were washed, FC blocked, surface stained forCD11c-APC, fixed and permeabilized for intracellular staining withanti-IL-10 FITC. Samples were analyzed by flow cytometry gating on CD11cpositive cells.

EXAMPLE 4 Regulation of IL-10 Production in Mature DCs ControlledThrough CD86 Exon 10

To further examine the question regarding the role of CD86 in theregulation of IL-10 production in maturing DCs we determined whatcomponents of the CD86 polypeptide might be responsible. The approachtaken was to systematically alter the CD86 protein by limitingexpression of either intracellular or extracellular polypeptide domains.This was done by using antisense oligomers with sequences targetingsplice sites within the unprocessed message thereby forcing alterationsin the mature message to exclude particular exons (Target and targetingsequences are shown in Table 1 and Table 2, respectively).

Employing RT-PCR on total RNA isolated from murine DCs treated with thedifferent oligomers shows that the predicted alterations to the CD86mRNA could be achieved (FIGS. 8A and 8B). Some cloned sequencesexhibited an alternative splice acceptor site in Exon 8 which has beenshown to be used in normal APCs (Borriello, Oliveros et al. 1995).

The data in FIGS. 8A and 8B demonstrate that antisense targeting ofsplice donor or splice acceptor sites alters CD86 mRNA. The data weregenerated using bone marrow derived DCs treated with [10 mM] of eitherP002-CD86 (SEQ ID NO:15) antisense PMO or scramble PMO peptideconjugates or P005 PMO peptide conjugates targeting splice junctionsites for exons 7, 9, 10 and 11 (SEQ ID NOS: 17, 18, 19 and 20,respectively) for 4 hours and then treated for 16 hours with LPS [1.0mg/ml]. Total RNA was isolated from each culture, treated with DNAsefree RNAse and used as template material for single-tube reversetranscription and polymerase chain reaction using primers spanning an870 base pair region on the CD86 mRNA. As shown in FIG. 8A, the reactionmaterial was fractionated on an EtBr stained 3.0% agarose gel todetermine the size of the resulting amplicons. FIG. 8B shows a schematicof the altered splicing patterns that were observed. The continuity ofthe open reading frames were confirmed by sequencing various clones ofthe amplicons after insertion into plasmid DNA vector. A schematicrepresentation of some of the cloned sequences is shown in FIG. 8B bylines aligned with the wild type CD86 map. The intervening black linesdepict regions where splicing was altered. The relative positions of thePMOs and the PCR primers are also shown.

EXAMPLE 5 Inhibition of CD86 or CD86 Exon 10 Expression AltersMorphology of LPS-Treated DCs

In addition to molecular evidence of the effect of the splice alteringoligomers as described in Example 4, phenotypic changes in the treatedDCs were also observed. Specifically, DCs treated with the Exon 10antisense oligomer (SEQ ID NO: 19) exhibited the greatest production ofIL-10 and maintained an immature phenotype when exposed to LPS (Table 4,below and FIG. 9, respectively). Table 4, shows that antisense PMOdesigned to block Exon 10 expression induces IL-10 in mature dendriticcells. FIG. 9 shows that inhibition of CD86 or Exon 10 expression altersthe morphology of LPS treated DCs. In this experiment, bone marrowderived DCs were treated as previously described in Example 4. Prior tostaining for flow cytometry the cultured DCs were imaged by lightmicroscopy. The forward and side light scattering properties of eachculture are shown to the right of each image. TABLE 4 Exon 10 of theCD86 Gene Regulates IL-10 production in Mature Dendritic Cells % DCs %DCs Treatment CD86 Positive IL10 Positive Control 10.40 0.30 Control (+LPS) 33.81 0.07 B7-2 AUG (SEQ ID NO: 15) + LPS 17.93 2.06 EXON 9sd (SEQID NO: 18) + LPS 26.62 1.48 EXON 10sa (SEQ ID NO: 19) + LPS 13.36 4.04EXON 11sa (SEQ ID NO: 20) + LPS 5.06 0.65

1. A method of inducing human dendritic cells to a condition of reduced capacity for antigen-specific activation of T cells, and, in mature dendritic cells, increased production of extracellular IL-10, comprising (a) exposing a population of human dendritic cells to a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to an expression-sensitive region of a preprocessed or processed human CD86 transcript identified, in its processed form, by SEQ ID NO:33, to form a heteroduplex structure between said compound and transcript having a Tm of at least 45° C., (b) by said forming, blocking expression of full-length CD86 in said cells, and (c) by said blocking, producing inhibition of the expression of full-length CD86 on the surface of dendritic cells, and enhanced expression of extracellular IL-10 by mature dendritic cells.
 2. The method of claim 1, wherein the antisense compound to which the dendritic cells are exposed is composed of phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.
 3. The method of claim 2, wherein the morpholino subunits in the compound to which the dendritic cells are exposed are joined by phosphorodiamidate linkages, in accordance with the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino, and said heteroduplex structure formed in step (a) has a Tm of at least 50° C.
 4. The method of claim 3, wherein X═NR₂, where each R is independently hydrogen or methyl in the compound to which the T cells are exposed.
 5. The method of claim 1, wherein the dendritic cells which are exposed to said compound include mature dendritic cells, and said blocking step (b) is effective to enhance expression of extracellular IL-10 by the dendritic cells.
 6. The method of claim 1, wherein said compound is covalently linked, at one compound end, to an arginine-rich peptide effective to enhance uptake of said compound into the dendritic cells.
 7. The method of claim 6, wherein said arginine-rich peptide has a sequence selected from the group consisting of SEQ ID NOS: 1 and
 2. 8. The method of claim 6, wherein said dendritic cells which are exposed to said compound include a mixture of immature and mature dendritic cells, said arginine-rich peptide is an rTAT peptide having the sequence identified by SEQ ID NO: 1, and the rTAT peptide is effective to achieve a greater level of intracellular uptake of the antisense compound into the mature dendritic cells than is achieved (i) in the immature dendritic cells, or (ii) by exposing the mature dendritic cells to the antisense compound in the absence of the rTAT polypeptide.
 9. The method of claim 1, wherein said antisense compound is effective to hybridize to a target region adjacent the start site of the processed human CD86 transcript, the compound has a base sequence that is complementary to a target region containing at least 12 contiguous bases in a processed human CD86 transcript identified by SEQ ID NO:9, and said blocking step (b) is effective to block translation of said processed transcript.
 10. The method of claim 9, wherein the antisense compound includes a base sequence selected from the group consisting of: SEQ ID NOS:21-23 and
 32. 11. The method of claim 1, wherein the antisense compound is effective to hybridize to a splice site of preprocessed human CD68, and has a base sequence that is complementary to at least 12 contiguous bases of a splice site in a preprocessed human CD86 transcript, and said blocking step (b) is effective to block processing of a preprocessed CD86 transcript to produce a full-length, processed CD86 transcript.
 12. The method of claim 11, wherein the splice site in the preprocessed CD86 transcript has a sequence selected from the group consisting of: SEQ ID NOS:10-14.
 13. The method of claim 12, wherein the antisense compound includes a base sequence selected from the group consisting of: SEQ ID NOS:24-28.
 14. The method of claim 1, for use in inhibiting transplantation rejection in a human subject receiving an allograft tissue or organ, wherein said exposing includes administering the antisense compound to the subject, in an amount effective to inhibit the rate and extent of rejection of the transplant.
 15. The method of claim 14, wherein said administering is carried out both prior to and following the allograft tissue or organ transplantation in the subject.
 16. The method of claim 14, wherein said administering is carried out for a selected period of 1-3 weeks.
 17. The method of claim 16, which further includes further administering the antisense compound to the subject, as needed, to control the extent of transplantation rejection in the subject.
 18. The method of claim 1, for use in treating an autoimmune condition in a human subject, wherein said exposing includes administering the antisense compound to the subject, in an amount effective to reduce the severity of the autoimmune condition.
 19. The method of claim 18, wherein said administering is carried out over an extended period of time, as needed, to control the severity of the autoimmune condition in the subject.
 20. A method of inducing mature human dendritic cells to a condition of increased production of extracellular IL-10, comprising (a) exposing the population of cells containing mature dendritic cells to a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to an expression-sensitive region of a preprocessed or processed human CD-86 transcript identified, in its processed form, by SEQ ID NO:33, to form a heteroduplex structure between said compound and transcript having a Tm of at least 45° C., (b) by said forming, blocking expression of full-length CD86 in said cells, and (c) by said blocking, enhancing expression of extracellular IL-10 by the mature dendritic cells.
 21. A composition for use in inducing dendritic cells to a condition of reduced capacity for antigen-specific activation of T cells, and, in mature dendritic cells, increased production of extracellular IL-10, said conjugate comprising, a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to an expression-sensitive region of a preprocessed or processed human CD-86 transcript identified, in its processed form, by SEQ ID NO:33, to form a heteroduplex structure between said compound and transcript having a Tm of at least 45° C.
 22. The composition of claim 21, wherein the antisense compound is composed of phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.
 23. The composition of claim 22, wherein the morpholino subunits in the compound to which the dendritic cells are exposed are joined by phosphorodiamidate linkages, in accordance with the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino, and said heteroduplex structure has a Tm of at least 50° C.
 24. The composition of claim 27, wherein X═NR₂, where each R is independently hydrogen or methyl in the compound to which the T cells are exposed.
 25. The composition of claim 21, which further includes an arginine-rich peptide covalently linked to one end of said antisense compound, and said peptide is effective to promote uptake of the composition into dendritic cells.
 26. The composition of claim 25, wherein said arginine-rich peptide is an rTAT peptide having the sequence identified as SEQ ID NO: 1, and said peptide is effective to achieve a greater level of intracellular uptake of the antisense compound into the mature dendritic cells than is achieved (i) in the immature dendritic cells, or (ii) by exposing the mature dendritic cells to the antisense compound in the absence of the rTAT polypeptide. 